HIV protease inhibitors, such as, ritonavir and saquinavir, are substrates for xenobiotic efflux pumps, e.g., P-glycoprotein and Mrp2 and thus penetrate the blood-brain barrier poorly. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice to validate signaling-based changes in blood-brain barrier transporter function in vivo. Our recent studies have focused on identifying signals that modify pump activity in the barrier. These in vitro and in vivo experiments with animal models suggest two specific strategies for modifying this barrier to improve drug delivery, but also potentially important complications of polypharmacy related to xenobiotic upregulation of efflux transporter expression. One strategy for improving CNS delivery of drugs that are P-glycoprotein substrates involves activating protein kinase C (PKC) isoform beta1 or sphingolipid signaling at the blood-brain barrier. This causes a rapid and reversible reduction in basal P-glycoprotein transport activity in isolated brain capillaries and in intact rats. We have now identified a drug, Fingolimod (FTY720) currently in use in the clinic, that will target this signaling system and reduced P-glycoprotein tranport activity in vitro and in vivo. In vivo, such signaling increases brain uptake of drugs that are P-glycoprotein substrates. Thus, targeting signals that regulate basal activity of this transporter increases delivery of therapeutic drugs to the brain, including HIV protease inhibitors. A second strategy to improve HIV drug delivery to the CNS involves use of an innovative chemical strategy with the P-gp substrate and anti-viral agent, abacavir, in conjunction with a traceless tether. Dimeric prodrugs of abacavir were designed to have two functions: inhibit P-gp efflux at the BBB and revert to monomeric therapeutic within cellular reducing environments. The prodrug dimers are potent P-gp inhibitors in cell culture and in a brain capillary model of the BBB. Significantly, these agents demonstrate anti-HIV activity in two T-cell-based HIV assays, a result that is linked to cellular reversion of the prodrug to abacavir. This strategy represents a platform technology that may be applied to other therapies with limited brain penetration due to P-glycoprotein.